PROJECT ABSTRACT: Fluorescence applications penetrate nearly every field of biological research. At all scales, investigations into biological systems rely heavily on the availability and performance of high-quantum yield fluorescent probes, most important and widespread of which are low molecular weight organic compounds that emit light in distinct, separable regions of the visible spectrum. Despite their demonstrated utility in serving as critical reagents in a myriad of diagnostic tools and advancing our understanding of biological mechanisms, the overall performance of organic fluorophores is limited by their photostability and phototoxicities in complex biological environments. Critically, the photon budgets of all fluorophore classes are limited by undesirable photophysical properties that limit both the photon flux as well as the total time interval over which photon emission events can be observed. Such phenomena, which include both transient (blinking) and irreversible (photobleaching) dark state excursions, add key uncertainties to all fluorescence applications. Issues of this nature are particularly limiting for single-molecule fluorescence studies, including super-resolution techniques, where relatively high levels of illumination intensity must be employed. During the current funding period, we have demonstrated the capacity to engineer and effectively utilize cyanine fluorophores spanning the visible spectrum that exhibit 3-100-fold increases in brightness and photostability, respectively. These critical advances have enhanced the time resolution and signal-to-noise ratio of single-molecule imaging investigations in both in vitro and in vivo settings to shed unprecedented functional insights on a variety of distinct biological systems that could not have been otherwise achieved. This increase in fluorophore performance directly correlates with reductions in photo-induced generation of reactive oxygen species that can impart harmful phototoxic effects in the biological systems in which they are utilized. Here we propose to build on the quantitative understanding of fluorophore performance established during the initial funding period to develop and implement predictably tunable intra-molecular photostabilization strategies. In so doing, we will generate a suite of chemically distinct organic fluorophores widely used in fluorescence applications that exhibit up to 200-fold increases in photostability across the visible spectrum. Such technologies are anticipated to revolutionize the investigation of biological processes in vitro, in living cells and ultimately in animals.